This proposed project is to elucidate a novel cellular DNA damage response network (DDR) pathway that inhibits the replication of polyomavirus (PyV) genomes. PyVs are small double-stranded DNA viruses that have high infection rates in their hosts (BK and JC viruses are present at levels as high as 80-90%). PyV infections don't cause appreciable disease in hosts with fully functional immune systems; however, in immuno- compromised hosts PyVs cause serious health problems (e.g. ? in kidney, bone-marrow, or multiple-organ transplant patients BK viremia can cause organ failure, and in HIV/AIDS patients JC can cause progressive multifocal leukoencephalopathy, a frequent cause of death in AIDS patients.) PyVs encode only a single viral enzyme, the PyV Large T-antigen (LT) the replicative DNA helicase, and to date no therapeutic agents exist that act directly against LT or PyVs. Developing a better understanding of PyV DNA replication and how that process can be inhibited, may provide avenues for the future development of anti-PyV antivirals. The DDR is a complex inter-connected and often redundant network of responses to DNA damage with a variety of effects on cells. DDR is a critical factor in how cancers arise and in many types of cancer treatments. Hence, better understanding of cellular DDR pathways is of the utmost importance to human health. It has been shown, by us as well as others, that activation of DDR can dramatically inhibit PyV DNA replication. PyV DNA replication can be arrested by the canonical DDR effects on cell cycle kinases. However, our preliminary results show that there is a second DDR pathway that inhibits PyV DNA replication through a non-canonical pathway. This novel pathway appears to act via direct phosphorylation of DNA replication proteins by the apical DDR kinases, the PIKKs. While cellular DNA replication proteins are known to be phosphorylated by PIKKs, little is known about the effects of these modifications. One of the replication proteins directly phosphorylated by the PIKKs, that appears to be mediating the arrest of PyV DNA replication is the PyV LT protein. The goals of the proposed research are to map the PIKK phosphorylation sites on PyV LT and the cellular replication proteins it interacts with, and to determine how DDR affects recruitment of LT and these replication proteins to PyV replicons (and whether this is associated with specific DDR- phosphorylation states of these proteins). Cell-based PyV DNA replication assays (for SV40, BK and JC PyVs) will be used to screen for compromised DNA replication function of specific phospho-site mutants of LT. PyV replication-compromised LT mutants will be expressed, purified, and analyzed for the various LT functions required to support PyV DNA replication. These studies will identify new ways for inhibiting PyV DNA replication by identifying and characterizing a novel cellular DDR pathway that affects PyV replication directly. This pathway between DDR and PyV DNA replication may also reflect and provide leads for investigating a hitherto unknown DDR pathway for regulating cellular DNA replication.